The effect of paraformaldehyde fixation and sucrose cryoprotection on metal concentration in murine neurological tissue

Abstract

Aldehyde preservation of tissue and cryoprotection by sugars and polyols has traditionally been used in histology to preserve structural integrity of samples. The effect of paraformaldehyde (PFA) fixation and sucrose cryoprotection on murine brain metals was studied by examining the changes in total brain metal levels throughout a complete fixation/cryoprotection protocol. Blank-corrected metal levels in 4% PFA and two changes of 30% sucrose solutions following brain immersion were determined by ICP-MS. Fixed and cryoprotected tissue was lyophilised, digested in HNO₃/H₂O₂ and analysed using the same ICP-MS protocol. The sum of metal loss or gain in each solution and total digest was used to determine the percentage of total brain metal present in fixed, cryoprotected tissue. Almost all K (99.78%) and Mg (78.01%) leached from brain tissue during fixation and cryoprotection, whilst Ca and Sr either retained or absorbed additional metal ions from the sucrose solution. Less water-soluble transition metals displayed smaller degrees of metal leaching, with Fe, Cu and Zn losing 26.61–31.62% of their total metal content during preparation for cryosectioning. These results suggest that metal leaching is highly metal specific, and that preparatory steps for cryosectioning of brain tissue should be approached according to this specificity.